There are two different general ways of determining the locations of molecules in a sample, which emit fluorescence light. According to the first general way, the locations are deduced from the spatial distribution of the fluorescence light registered by means of a light sensor array. According to the second general way, the locations of the molecules are assigned to spatially delimited excitation areas in which the molecules are locally excited for the emission of the fluorescence light and for which the fluorescence light emitted by the molecules is registered separately.
According to the first general way in which the fluorescence light emitted by the substance is registered by means of a light sensor array onto which the sample is imaged by means of an objective lens, the spatial resolution or accuracy achievable in imaging the distribution of the molecules of the substance in the sample or in imaging a structure of the sample marked with the substance is delimited by the so-called Abbe diffraction limit. According to the Abbe diffraction limit, one may determine a location of a molecule in a sample emitting an individual photon which has been registered at a certain location of the light sensor array only at a spatial uncertainty in the region of λ/2n sin α, wherein λ is the wavelength of the fluorescence light, n is the refraction index of the optical material arranged between the sample and the objective lens, and α is half the aperture angle of the objective lens.
If, however, the fluorescence light registered at the light sensor array may be assigned to a single fluorescing molecule in the sample and if the single fluorescing molecule emits a higher number of photons, the location of the molecule can be determined at a spatial accuracy beyond the diffraction limit. For this purpose, the center of the distribution of the positions of the light is sensor array is determined at which the individual photons are registered, and the location of the emitting molecule is deduced therefrom. The spatial resolution achieved by this method which is called localization increases with √n, wherein n is the number of the photons emitted by the molecule and registered by the light sensor array.
If a molecule emits fluorescence light in a directional spatial distribution, this results in a spatial error in determining the location of the molecule from the intensity distribution of the fluorescence light over a light sensor array by means of localization. This spatial error depends on the orientation of the molecule within the sample. A directional spatial distribution of the emitted fluorescence light is, for example, displayed by molecules whose rotation diffusion times are longer than the lifetimes of their excited state out of which they emit the fluorescence light. (See Engelhardt, J. et al., Molecular orientation affects localization accuracy in superresolution far-field fluorescence microscopy, Nano Lett. 2011, Jan. 12; 11(1): 209-13.)
International Patent Application publication WO 2006/127692 discloses to mark a structure of interest in a sample with molecules of a substance which are in a non-fluorescent starting state but which, by means of adjusting light, can be switched into a fluorescent state. Thus, by means of the adjusting light, a small proportion of the molecules can be brought into the fluorescent state in which the distances to nearest neighboring molecules in the fluorescent state are greater than the diffraction limit. With a following application of excitation light, only the molecules in the fluorescent state emit fluorescence light. Thus, the fluorescence light from the individual molecules in the fluorescent state can be registered separately, and the locations of the individual molecules may be determined by means of localization at a spatial accuracy beyond the diffraction limit despite the high absolute concentration of the molecules in the substance. Imaging the distribution of the molecules of he substance in the sample is achieved successively in that the steps of transferring a small proportion of the molecules into the fluorescent state, of exciting these molecules for the emission of fluorescence light, and of registering the fluorescence light by means of a light sensor array are repeated and, thus, for statistical reasons, executed for different portions of the molecules of the substance in the sample.
WO 2006/127692 also describes that activating a portion of the molecules of the substance into a fluorescent state may be combined with other optical imaging methods. For example, by means of an intensity distribution of the excitation light comprising maxima delimited by minima, the molecules of the substance may only be excited for the emission of fluorescence light in certain planes or other spatial subunits of the sample.
The method known from WO 2006/127692 for determining the position of each molecule of the substance at a spatial accuracy beyond the diffraction limit requires a number of photons from the molecule, and this number increases with the desired spatial accuracy. Further, the method makes great demands on the molecules to enable that only a small proportion of the molecules can be switched into the fluorescent state, the distances between the molecules switched into the fluorescent state being greater than the diffraction limit. Typically, the molecules are photochromic fluorophores or proteins which can be switched between two conformation states one of which being fluorescent.
The method known from WO 2006/127692 is also known as PALM, i.e. Photo-Activated Localization Microscopy. A similar method known as STORM (Stochastic Optical Reconstruction Microscopy) basically has the same advantages and drawbacks.
U.S. Pat. No. 8,174,692 B2 discloses that even standard dyes which are not switchable but have a fluorescent starting state, which are arranged at smaller distances than the diffraction limit, and which may not be switched between two conformation states, only one of which being fluorescent, can be used as a substance to determine the location of individual molecules of the substance by means of localization. Here, the sample is subjected to excitation light, which, at a certain transition probability, transfers the molecules into a relatively long-living electronic dark state, at such a high intensity that the molecules still being in the fluorescent starting state are arranged at distances beyond the diffraction limit.
By means of the excitation light, the molecules presently being in their fluorescent state are excited for the emission of fluorescence light which is registered with spatial resolution by means of a light sensor array. In this way, successively, different molecules of the substance are localized, as the molecules from which photons have already been registered get into the dark state, whereas other molecules, at a certain transition probability, return into the fluorescent state. This known method can be carried out continuously, i.e. frames may be continuously read out of the light sensor array whereas the sample is subjected to a high intensity of the excitation light which essentially keeps the substance in its dark state and only excites isolated molecules for the emission of fluorescence light.
The method known from U.S. Pat. No. 8,174,692 B2 is also designated as GSDIM (Ground State Depletion Individual Molecule Return Microscopy).
The high spatial resolution in determining the locations of isolated or individual fluorescent molecules by means of localization is only achieved in the x- and y-direction running orthogonal to the optical axis but not in the z-direction of the optical axis of the objective lens by which the respective sample is imaged onto the light sensor array. However, from Aquino, D. et al., Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores, Nature Meth. 8,353-359 (2011) it is known that the locations of the individual fluorescence molecules in a PALM-, STORM-, or GSDIM-method can be determined by means of a 4Pi method with two facing objectives aligned in the z-direction of the optical axis by scanning the sample with an x-y-measurement plane.
When the locations of molecules emitting fluorescence light are equated with the position of their spatially limited excitation for emission of fluorescence light, the respective sample being scanned or rasterized with the locations of their spatially limited excitation, this is called scanning fluorescence light microscopy. With regard to the spatial resolution or accuracy achieved in scanning fluorescence light microscopy, the Abbe diffraction limit also normally applies, here at the wavelength of the excitation light. However, some methods are known by which the spatial accuracy in scanning fluorescence light microscopy can be enhanced beyond the diffraction limit by means of reducing the effective spatial area of the excitation of the molecules of the substance for emission of fluorescence light.
In scanning fluorescence light microscopy, the registration of any photons is sufficient to determine whether molecules of the substance emitting fluorescence light are located in the present area of the excitation. The number of the photons registered is only used for determining the local concentration of the molecules of the substance in the present area of the excitation.
In STED (Stimulated Emission Depletion) fluorescence light microscopy, directly after molecules of a substance marking a structure of a sample have been excited by means of excitation light, the excitation is removed in surroundings of a measurement point of interest by means of directed emission. This directed emission is stimulated by means of STED light and inhibits the emission of fluorescence light by the molecules so that the fluorescence light may only come out of that area of the sample in which the excitation has not been removed. The area in which the excitation has not been removed can be kept very small in that it is defined by means of a zero point or null of the intensity distribution of the STED light and in that the absolute intensity of the STED light is adjusted so high that it completely removes the excitation of the molecules even very close to the zero point.
Instead of removing a previously effected excitation of the molecules in parts of the sample, light having an intensity distribution comprising a zero point may also be used for switching the molecules of the substance into a non-fluorescent conformation state, like it is done in RESOLFT fluorescence light microscopy, or into an electronic dark state, like it is done in GSD (Ground State Depletion) fluorescence light microscopy.
U.S. Pat. No. 7,485,875 B2, corresponding to DE 10 2005 034 443 A1, discloses a GSD method using light of one wavelength only. Up to a certain intensity, this light excites the substance primarily for the emission of fluorescence light. Above this intensity, the light transfers the molecules of the substance essentially completely into a dark state. In that an intensity distribution, at which the light is applied to the sample, has a local minimum lower than the certain intensity described, the area in which the molecules of the substance are effectively excited for the emission of fluorescence light is spatially delimited.
International Patent Application publication WO 2012/171999 teaches to scan a sample with a beam of excitation light surrounded by an intensity distribution of STED light having a minimum at the focus point of the excitation light so quickly that, in each of several scanning passages, the fluorescence light registered consists of individual photons emitted by individual molecules. The locations of the molecules are equated with the positions of the focus point of the beam of excitation light at which the photons of the fluorescence light has been registered.
U.S. Pat. No. 9,291,562 B2, corresponding to DE 10 2011 055 367 A1, discloses a method of tracking a single fluorescent molecule, which, by means of excitation light, is caused for the emission of fluorescence light, the fluorescence light being registered. The excitation light is applied to the sample with an intensity distribution comprising a local minimum, and the molecule moving within the sample is tracked with the local minimum. For this purpose, the intensity distribution of the excitation light is shifted moved with regard to the sample such that an intensity of the fluorescence light emitted by the particle remains minimal. The minimal intensity of the fluorescence light emitted by the particle is that rate at which individual photons are emitted by the respective molecule if the molecule is located in the minimum of the intensity distribution of the excitation light. This minimum may be a zero point or null of the intensity distribution of the excitation light.
U.S. Pat. No. 9,024,279 B2, corresponding to DE 10 2010 028 138 A1, discloses a method of determining the distribution of a substance in a measurement area by means of scanning with a measurement front. Over a depth of the measurement front which is shorter than the diffraction limit at the wavelength of an optical signal, the intensity of the optical signal increases in such a way that a proportion of the substance in a measurement state at first increases from non-existing and then drops down to non-existing again. The measurement front is shifted or moved over the measurement area in opposite direction to the increase of the intensity of the optical signal. The measurement signal emitted out of the area of the measurement front is registered and assigned to the respective position of the measurement front within the measurement area.
In so-called SSIM (Saturated Structured Illumination Microscopy, see Gustafsson, M. G. L., Proc. Natl. Acad. Sci. USA 102, 13081-13086 (2005)), a sample is scanned in different directions with an intensity distribution of excitation light which has a line-shaped zero point and such high intensities outside the zero point that a saturation of the intensity of the fluorescence light by excited molecules of a fluorescent substance within the sample is achieved. Fluorescence light from the sample registered during scanning varies due to the fact that molecules of the fluorescent substance which are presently in the area of the zero point do not contribute to the fluorescence light. This fluorescence light is evaluated with regard to spatial frequencies which develop in scanning in the various directions and from which an image of the distribution of the fluorescent molecules in the sample may then be reconstructed.
SSIM does not make use of a distribution of the fluorescent molecules in the sample in which an average distance of the molecules is higher than the diffraction limit at the wavelength of the excitation light or the fluorescence light. SSIM may only be carried out with fluorescent molecules which can be excited up to the saturation of the intensity of the fluorescence light emitted by them without transferring them into a dark state. The image of the distribution of the molecules of the fluorescent substance in the sample is only achieved indirectly. Positions of individual fluorescent molecules in the sample are not determined.
There still is a need of a method and an apparatus for determining the locations of individual molecules of a substance in a sample by which a high spatial resolution image of a distribution of the molecules of the substance in the sample can be produced by evaluating the fluorescence light obtained from the individual molecules, which may additionally be used for determining the locations of individual molecules by means of localization.